The present invention relates to the novel use of compounds that disrupt tight junctions to facilitate the intracellular delivery (and/or transfection) of biologically active molecules by lipids, cationic amphiphilic compounds, non-viral and viral vectors.
The effective introduction of foreign genes and other biologically active molecules into targeted mammalian cells is a challenge still facing those skilled in the art. Gene therapy requires successful transfection of target cells in a patient. Transfection, which is practically useful per se, may generally be defined as a process of introducing an expressible polynucleotide (for example a gene, a cDNA, or an mRNA) into a cell. Successful expression of the encoding polynucleotide thus transfected leads to production in the cells of a normal protein and is also practically useful per se. A goal, of course, is to obtain expression sufficient to lead to correction of the disease state associated with the abnormal gene.
Examples of diseases that are targets of gene therapy include: inherited disorders such as cystic fibrosis, Gaucher""s disease, Fabry""s disease, and muscular dystrophy. Representative of acquired target disorders are: (1) for cancersxe2x80x94multiple myeloma, leukemias, melanomas, ovarian carcinoma and small cell lung cancer; (2) for cardiovascular conditionsxe2x80x94progressive heart failure, restenosis, and hemophilias; and (3) for neurological conditionsxe2x80x94traumatic brain injury.
Cystic fibrosis, a common lethal genetic disorder, is a particular example of a disease that is a target for gene therapy. The disease is caused by the presence of one or more mutations in the gene that encodes a protein known as cystic fibrosis transmembrane conductance regulator (xe2x80x9cCFTRxe2x80x9d). Cystic fibrosis is characterized by chronic sputum production, recurrent infections and lung destruction (Boat, T. F., McGraw-Hill, Inc., 1989, p. 2649-2680). Though it is not precisely known how the mutation of the CFTR gene leads to the clinical manifestation (Welsh, M. J. et al. Cell 73:1251-1254, 1993), defective Cl secretion and increased Na+ absorption (Welsh, M. J. et al., Cell 73:1251-1254, 1993; Quinton, P. M., FASEB Lett. 4:2709-2717,1990) are well documented. Furthermore, these changes in ion transport produce alterations in fluid transport across surface and gland epithelia (Jiang, C. et al., Science 262:424-427, 1993; Jiang, C. et al., J. Physiol. (London), 501.3:637-647, 1997; Smith, J. J. et al. J. Clin. Invest, 91:1148-1153, 1993; and Zhang, Y. et al., Am.J.Physiol 270:C1326-1335, 1996). These resultant alterations in water and salt content of airway liquid (ASL) may diminish the activity of bactericidal peptides secreted from the epithelial cells (Smith, J. J. et al., Cell, 85:229-236, 1996) and/or impair mucociliary clearance, thereby promoting recurrent lung infection and inflammation.
It is widely expected that gene therapy will provide a long lasting and predictable form of therapy for certain disease states such as CF, however, there is a need to develop improved methods that facilitate entry of functional genes into cells, and whose activity in this regard is sufficient to provide for in vivo delivery of genes or other such biologically active molecules.
Effective introduction of many types of biologically active molecules has been difficult and not all the methods that have been developed are able to effectuate efficient delivery of adequate amounts of the desired molecules into the targeted cells. The complex structure, behavior, and environment presented by an intact tissue that is targeted for intracellular delivery of biologically active molecules often interfere substantially with such delivery. Numerous methods and delivery vehicles including viral vectors, DNA encapsulated in liposomes, lipid delivery vehicles, and naked DNA have been employed to effectuate the delivery of DNA into the cells of mammals. To date, delivery of DNA in vitro, ex vivo, and in vivo has been demonstrated using many of the aforementioned methods.
Though viral transfection is relatively efficient, the host immune response frequently poses a major problem. Specifically, viral proteins may activate cytotoxicity T lymphocytes (CTLs) which destroy the virus-infected cells thereby terminating gene expression in the lungs of in vivo models examined. The other problem is diminished gene transfer upon repeated administration of viral vectors due to the development of antiviral neutralizing antibodies. These issues are presently being addressed by modifying both the vectors and the host immune system, however, a more efficient method of viral transfection or delivery is also desirable.
For example, the relatively low efficacy of AdV mediated gene transfer to airway epithelial cells is a major barrier for gene therapy of CF. Gene therapy with recombinant adenoviral (AdV) vectors may also lead to inflammatory and immune responses. For applications in which repeat therapy is necessary, such as CF, these responses can limit the therapeutic usefulness of the vector. In principle, the utility of vectors may be improved by increasing its therapeutic index, i.e., by either increasing its efficacy or decreasing its toxicity. For example, a strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given amount of transgene expression, and thereby also reduce unwanted effects such as immune responses.
Gene transfer using AdV has been proposed as a method to treat CF. Although the ability of AdV vectors to deliver the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells has been demonstrated by many laboratories, this process has been shown to be relatively inefficient. In addition, while treatment with AdV expressing CFTR has been shown to correct the chloride channel defect in human CIF airway epithelia grown in culture, the ability to correct the enhanced sodium absorption exhibited by these cells has been much less apparent. As a result of this inefficiency, relatively high doses of AdV need to be administered in vivo to observe significant correction of physiologic deficits. Due to the undesirable host immune response associated with delivering high doses of AdV, it is desirable that both the transfer efficiency and level of expression from AdV be improved to develop an effective treatment for CF.
Additionally, non-viral and non-proteinaceous vectors have been gaining attention as alternative approaches. Because compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both non-polar and polar environments (in or on, for example, the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself, such compounds are designed typically to contain both polar and non-polar domains. Compounds having both such domains may be termed amphiphiles, and many lipids and synthetic lipids that have been disclosed for use in facilitating such intracellular delivery (whether for in vitro or in vivo application) meet this definition. One group of amphiphilic compounds that have showed particular promise for efficient delivery of biologically active molecules are cationic amphiphiles. Cationic amphiphiles have polar groups that are capable of being positively charged at or around physiological pH, and this property is understood in the art to be important in defining how the amphiphiles interact with the many types of biologically active molecules including, for example, negatively charged polynucleotides such as DNA.
Several recently issued U.S. patents, the disclosures of which are specifically incorporated by reference herein, have described the utility of cationic amphiphiles to deliver polynucleotides to mammalian cells. (U.S. Pat. No. 5,676,954 to Brigham et al. and U.S. Pat. No. 5,703,055 to Felgner et al.)
Although the compounds mentioned in the above-identified references have been demonstrated to facilitate the entry of biologically active molecules into cells, it is believed that the uptake efficiencies provided thereby could be improved to support numerous therapeutic applications, particularly gene therapy. Additionally, it is sought to improve the activity of the above-identified compounds so that lesser quantities thereof are necessary, leading to reduced concerns about the toxicity of such compounds or of the metabolites thereof.
Another class of cationic amphiphiles with enhanced activity is described, for example, in U.S. Pat. No. 5,747,471 to Siegel et al. issued May 5, 1998, U.S. Pat. No. 5,650,096 to Harris et al. issued Jul. 22, 1997, and PCT publication WO 98/02191 published Jan. 22, 1998, the disclosures of which are specifically incorporated by reference herein. These patents also disclose formulations of cationic amphiphiles of relevance to the practice of the present invention.
Another contributing factor to the lack of efficient intracellular delivery of biologically active molecules is the inherent difficulties in binding to the apical membrane of polarized epithelial cells. Epithelial cells are one of the most common cell types found in animals. They form boundaries between different compartments within the body and line the cavities of all the major organ systems. The major function of epithelial cells is to provide protection and to regulate transport of ions, small molecules and fluid. Epithelial cells acquire and maintain a polarized organization with respect to the membrane in order to facilitate their role as a transport regulator. Subsequently, the well-differentiated airway epithelium is the principal target tissue for gene transfer for the treatment of CF.
The plasma membrane of polarized epithelial cells is divided into apical and basolateral domains. The apical surface faces the outside environment and is specific to polarized epithelial cells. The basolateral epithelial surface has many features in common with non-epithelial cells and its major function is cell-cell and cell-substrate adhesion. These two regions of the membrane are divided by tight junctions. The tight junctions are thought to prevent uncontrolled passage of molecules across the epithelium and to help maintain a barrier between the apical and basolateral regions. Subsequently, the permeability of many therapeutic drugs is limited because of access to mainly the apical membrane.
One method of improving the permeability of therapeutic drugs is to utilize a group of compounds well known in the art which disrupt tight junctions (Adjei et al., Pharm. Res., 9, pp. 244-249 (1992); Freeman and Niven, Pharm. Res., 13, pp. 202-209 (1996); Tomita et al., J. of Pharm. Sciences, 85, pp. 608-611 (1996); and Tomita et al. J. of Pharm. and Exp. Therap., 261, pp. 25-31 (1992)). The mechanism of action of all tight junction disrupting compounds is not fully understood but the fundamental idea is that the passageways between cells are widened, thus enabling larger molecules to access the basolateral region or to cross the apical membrane into another region of the body. EDTA, for example, widens the paracellular routes via calcium chelation creating access to non-apical or basolateral membranes. Other tight junction disrupting compounds increase the intracellular calcium level by an interaction with the cell membrane, also widening paracellular routes. Use of tight junction disrupting compounds has been shown to greatly improve the permeability and absorption of numerous therapeutic drugs in mammals.
Compounds capable of preventing the formation of cell-cell adhesion junctions may also improve the permeability and adsorption of therapeutic drugs. For example, HAV peptides are a series of peptides containing the sequence of histidine, alanine and valine that modulate cadherin-mediated cell adhesion.
The present invention provides for a method of improving gene therapy by treating cells with a compound capable of disrupting the tight junctions of polarized epithelial cells. The tight junction disrupting compounds increase the uptake and binding to mammalian cells of compositions employed in the art to effectuate delivery of biologically active molecules, including cationic amphiphiles and other lipids and non-viral and viral vectors. The tight junction disrupting compounds also increase transfection of biologically active molecules to epithelial cells.
The invention provides for the use of tight junction disrupting compounds that are effective for both lipid and non-lipid methods of delivering biologically active molecules including non-viral and viral vectors. The invention provides for use of tight junction disrupting compounds with all lipid complexes, along with the use of tight junction disrupting compounds with non-viral and viral vectors including adenoviruses and other methods that have been employed in the art to effectuate delivery of biologically active molecules into the cells of mammals.
In a preferred embodiment, instilling EGTA may significantly enhance the expression from a subsequent administration of adenovirus. This effect of EGTA may also enhance the ability of a subsequent aerosol of adenovirus to transfect epithelial cells. Not to be limited as to theory, the enhancing effect of EGTA may result from its ability to open epithelial tight junctions. This pretreatment strategy may improve the therapeutic index of adenovirus for applications such as CF that involve transfection of airway epithelial cells.
In a further embodiment, instilling EGTA may significantly enhance the expression from a subsequent administration of a lipid composition comprising a biologically active molecule. More preferably, the lipid composition comprises a cationic lipid or cationic amphiphile. This effect of EGTA may also enhance the ability of a subsequent aerosol of a lipid composition to transfect epithelial cells. A tight junction disrupting pretreatment strategy may improve the therapeutic index of lipid composition for applications such as CF that involve transfection of airway epithelial cells.
In another aspect, the invention provides for pharmaceutical compositions of tight junction disrupting compounds and pharmaceutical compositions of lipid and non-lipid compositions with tight junction disrupting compounds. The tight junction disrupting compounds may be an active ingredient in a pharmaceutical composition that includes carriers, fillers, extenders, dispersants, creams, gels, solutions and other excipients that are common in the pharmaceutical formulatory arts.
In a preferred embodiment, the invention provides for a method of providing gene therapy by administering a composition of a compound capable of disrupting tight junctions and a pharmaceutical composition comprising other formulations that have been employed in the art to effectuate delivery of biologically active molecules into the cells of mammals. A preferred method of administration is aerosolization.
The invention also provides for pharmaceutical compositions that comprise one or more cationic amphiphiles or adenoviruses, and one or more biologically active molecules, wherein said compositions facilitate intracellular delivery and more preferably intracellular delivery in the tissues of patients of therapeutically effective amounts of the biologically active molecules. The pharmaceutical compositions of the invention may be formulated to contain one or more additional physiologically acceptable substances that stabilize the compositions for storage and/or contribute to the successful intracellular delivery of the biologically active molecules, such as tight junction disrupting compounds.
For pharmaceutical use, the cationic amphiphile(s) of the invention may be formulated with one or more additional cationic amphiphiles including those known in the art, or with neutral co-lipids such as dioleoylphosphatidyl-ethanolamine, (xe2x80x9cDOPExe2x80x9d), to facilitate delivery to cells of the biologically active molecules. Additionally, the cationic amphiphiles, and nonviral and viral vectors may be formulated with a targeting agent or a lipid to coat the composition in order to facilitate delivery of a biologically active molecule to a targeted cell or tissue.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by the practice of the invention. The objectives and other advantages of the invention will be realized and attained by the compounds and methods particularly pointed out in the written description and claims hereof as well as the appended drawings.